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P Bagnarelli

Publications and source records attributed to P Bagnarelli.

At least 19 recordsLinked to original sources
PubMed2004

Intra-host evolution of human immunodeficiency virus type 1 and viral fitness.

RNA viruses are frequently tolerant to high levels of mutagenesis. In contrast, DNA viruses are less errorprone and coevolve along with their specific hosts over long time periods. Although both strategies have been successful, the "RNA-strategy" (directly linked to the pathogenic potential of these agents) most often generates novelty (new variants, new strains, and even new viral pathogens). For several decades, intra-host virus evolution has been considered to be a speculative field, far from the main issues of clinical virology. This concept is now changed, due to the evidence that RNA virus evolution is intimately linked to failures in viral disease control and prevention. Antiviral strategies using single and fixed elements (i.e. treatments using one antiviral compound, immunizations using a single recombinant protein) have been unable to control highly dynamic quasispecies, such as human immunodeficiency virus type I (HIV-1) and hepatitis C virus (HCV). The development of combinatorial treatments in HIV-1 infection and the recognition that vaccines should be multivalent are important steps in adapting disease control strategies to the complexity of viral populations. The present report summarizes the strategies adopted to address HIV-1 evolution and its phenotypic consequences, including changes in susceptibility to antiviral compounds, viral fitness, and pathogenic potential. In particular, it is highlighted that sequence-function analyses of the intra-host HIV-I evolution, including studies of viral fitness, have opened up new perspectives not only to studying the pathogenic mechanisms and the virus-host relationships, but also to designing new strategies for monitoring antiviral therapies.

AIDS Vaccines↗
PubMed2004

Resistance and replicative capacity of HIV-1 strains selected in vivo by long-term enfuvirtide treatment.

Enfuvirtide is the prototype member of a new class of anti HIV-1 agents, the fusion inhibitors (FI). In recent clinical trials, the compound has shown its efficacy in combination with other antiretroviral agents in vivo. However mutant strains resistant to the action of the drug arise quite rapidly in vitro and in vivo. To analyze the process of selection and evolution of HIV-1 strains resistant to enfuvirtide in vivo and to evaluate the impact of resistance on viral fitness, 12 HIV-1 infected subjects treated with T20 (enfuvirtide) for at least one year were included in the study. Gp41-coding sequences were amplified from plasma samples of these subjects at baseline and at different time points during treatment. Seven of the 12 subjects showed selection of gp41 mutations under the selective pressure of enfuvirtide. In particular, these mutations clustered in two distinct regions: (i) a mutational hot-spot localized, as previously described, in the first residues of the N-HR domain, with position number 38 as the most heavily mutated, but including also a G36V, a N42D/T, a N43D, a L44M and a L45M; (ii) other mutations were localized further downstream, within N-HR/C-HR junction and in the C-HR. A recombinant assay specifically designed for the determination of HIV-1 phenotype to FI was developed and validated. Using this assay, we observed that all of the 7 mutated clones displayed substantially reduced susceptibility to T20, IC50 ranging from 0.6 to12.8 microg/ml (>100 fold change). The residues whose mutation was associated with a potent reduction in susceptibility were V38, N42, and N43, other positions such as G36, N44 and L45 playing a minor role. None of the mutant HIV isolates showed cross-resistance to T-1249. By the same method, the HIV-1 replicative capacity of the recombinant clones was tested in the absence of drugs, and for each subject, pre-therapy clones were compared to post-therapy ones. In 3/7 subjects a significant decrease in replicative capacity of the recombinant clones was observed. The phenotypic data from this study suggest that the secondary additional mutations, could be associated with improved resistance or recovery of replicative capacity (compensatory mutations).

Amino Acid Sequence↗
PubMed2004

Immune reconstitution in HIV-1 infected children with discordant response to antiretroviral therapies: patterns of HIV-1 env gene evolution driven by restoration of thymic function.

The evolution of human immunodeficiency virus type 1 (HIV-1) env gp120 region was addressed in HIV-1 infected children showing virological failure to antiretroviral therapy (ART). Sequence analysis of the replicating plasma virus at baseline and after one year of therapy documented evolution of gp120 in all subjects but one. Analysis of the host's selective pressure showed that the values of Ka/Ks ratios were higher in the V3 sequence than in the whole C2-V5 region in 4 of 5 children with improvement of thymic output. Moreover, in 2 of the 4 chidren, the V3 evolution paralleled with a reverse shift of the viral phenotype (from CXCR4-tropic to CCR5-tropic). These results suggest that, under ART, the V3 evolution towards less pathogenic viral variants may be driven by the host's increased selective pressure following restoration of thymic function and immune reconstitution.

Amino Acid Sequence↗
PubMed2000

Prevalence of multiple dideoxynucleoside analogue resistance (MddNR) in a multicenter cohort of HIV-1-infected Italian patients with virologic failure.

We evaluated the prevalence of both Q151M and 6-bp insert at position 69 of RT region responsible for multiple dideoxynucleoside analogue-resistant (MddNR) HIV-1 variants in 177 patients who failed to respond to combination therapy. Patients had received protease inhibitors (PI) and/or nonnucleoside reverse transcriptase inhibitors (NNRTIs) after a long-term experience with nucleoside reverse transcriptase inhibitors (NRTIs) (including zidovudine monotherapy). Two of 177 patients (1.1%) showed the specific complex of Q151M mutation, while 4 (2.3%) had the 69 6-bp insert. Mutations that belong to the 151 set in the absence of the pivotal Q151M substitution were detected in as many as 3.9% of the patients. One patient exhibited a 69S [VG] insert that has not been previously phenotypically characterized. This HIV-1 isolate had high levels of resistance to all NRTIs except stavudine. MddNR is an emerging problem after sequential therapy with this class of compounds among HIV-1-infected patients. Either didanosine (ddI) or zidovudine (ZDV) monotherapy allowed the emergence of MddNR variants containing Q151M complex. Monotherapy with ZDV and ddI or subsequent treatments with various NRTI combinations were the common background in the patients with the 69 insert. The overall prevalence of MddNR (3.4%) in Italy is comparable with that observed in several other European countries (3.4%-6.5%). These data suggest that patients failed by NRTI regimens should be analyzed for the presence of both patterns of MddNR.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Quantitative evaluation of the recombinant HIV-1 phenotype to protease inhibitors by a single-step strategy.

OBJECTIVE: To develop and optimize a fast and quantitative recombinant strategy for evaluating the HIV-1 phenotype to protease inhibitors (PI). DESIGN AND METHODS: A non-replicative HIV-1 molecular vector (designated pdelta prodelta env) capable of expressing exogenous HIV-1 protease-encoding sequences was developed in this study. The HIV-1 protease sequences were amplified from either viral isolates or plasma samples (both from 21 HIV-1-infected individuals, 19 of whom were failing different anti-HIV-1 combination treatments) and cloned in the pdelta prodelta env backbone. The HIV-1 recombinant phenotype to PI was determined directly after transfection of viral chimeric clones by measuring protease activity and calculating a percentage sensitivity index (SI%; the ratio between the results from each clone and those from a PI-sensitive reference strain). RESULTS: The SI% values obtained from the recombinant clones paralleled the IC50 results of the viral isolates and documented different degrees of resistance and cross-resistance to PI, compatible, with few exceptions, with the respective genotype. Interestingly, an inverse correlation between SI% values and the presence of primary mutations for resistance to PI (P = 0.0038 and P = 0.0414, for indinavir and ritonavir, respectively) and a difference in SI% between samples harbouring an increasing number of mutations (indinavir, P = 0.022; ritonavir, P = 0.0466) were observed. CONCLUSION: The data substantiate the reliability of the novel strategy for a fast (5 day) quantitative evaluation of HIV-1 phenotype to PI, and indicate that this method may contribute to the understanding of mechanisms of virus resistance to PI.

Chimera↗
PubMed1999

Defective nef alleles in a cohort of hemophiliacs with progressing and nonprogressing HIV-1 infection.

Deletion of the nef gene results in viral attenuation and confers protection against challenge with wild-type simian immunodeficiency virus in macaques. Regarding HIV-1 infection, a few long-term nonprogressors (LTNP) with nef deletions have been described. In this study, the nef genes of a group of seven LTNP and eight progressors, all belonging to the same cohort of infected hemophiliacs, were analyzed by cloning and sequencing from both virion RNA and peripheral blood mononuclear cell-associated proviral DNA. Defective nef sequences coexisted with full-length nef open reading frames in five of seven LTNP and two of eight progressors. The proportion of disrupted nef sequences within each individual was significantly higher in LTNP (ranging from 10 to 63%) than in progressors (ranging from 9 to 21%) (P = 0.013). Moreover, in-frame small deletions predicting to encode Nef were found in all RNA- and DNA-derived clones from one LTNP and four progressors. A chimeric virus in which the nef gene of NL4.3 was substituted with the nef allele containing the deletion of two alanines at position 49-50 found in two progressors showed a defective replicative capacity compared to NL4.3 virus. In summary, hemophiliacs with either progressing or nonprogressing HIV-1 infection are characterized by the presence of defective nef variants.

Adolescent↗
PubMed1999

Identification of two distinct subsets of long-term nonprogressors with divergent viral activity by stromal-derived factor 1 chemokine gene polymorphism analysis.

Stromal-derived factor (SDF)-1, the natural ligand for CXCR4, is present in a common polymorphic variant defined by a G-->A transition in the 3' untranslated region of the gene. In persons infected with human immunodeficiency virus type 1 (HIV-1), the homozygous genotype (SDF1-3'A/3'A) has been postulated to interfere with the appearance of T-tropic syncytium-inducing strains. The polymorphism of SDF1 was correlated with HIV-1 phenotype, plasma viremia, and unspliced and multiply spliced specific transcripts in 158 virologically characterized HIV-1-infected patients (39 recent seroconverters, 75 typical progressors, and 44 AIDS patients) and in 42 HIV-1-infected long-term nonprogressors (LTNPs). Analysis of SDF1 allele distribution revealed that SDF1-3'A/3'A status is associated with low CD4 cell count (P=.0449) but not with a specific HIV-1 phenotype. In LTNPs, SDF1-+/+ condition defined a subset of persons with lower HIV-1 replication than in heterozygous subjects. The low viral activity in SDF1-+/+ LTNPs suggests that other factors play a major role in vivo in determining the course of HIV-1 infection.

Adult↗
PubMed1999

Host-specific modulation of the selective constraints driving human immunodeficiency virus type 1 env gene evolution.

To address the evolution of human immunodeficiency virus type 1 (HIV-1) within a single host, we analyzed the HIV-1 C2-V5 env regions of both cell-free genomic-RNA- and proviral-DNA-derived clones. Sequential samples were collected over a period of 3 years from six untreated subjects (three typical progressors [TPs] and three slow progressors [SPs], all with a comparable length of infection except one. The evolutionary analysis of the C2-V5 env sequences performed on 506 molecular clones (253 RNA- and 253 DNA-derived sequences) highlighted a series of differences between TPs and SPs. In particular, (i) clonal sequences from SPs (DNA and RNA) showed lower nucleotide similarity than those from TPs (P = 0. 0001), (ii) DNA clones from SPs showed higher intra- and intersample nucleotide divergence than those from TPs (P < 0.05), (iii) higher host-selective pressure was generally detectable in SPs (DNA and RNA sequences), and (iv) the increase in the genetic distance of DNA and RNA sequences over time was paralleled by an increase in both synonymous (Ks) and nonsynonymous (Ka) substitutions in TPs but only in nonsynonymous substitutions in SPs. Several individual peculiarities of the HIV-1 evolutionary dynamics emerged when the V3, V4, and V5 env regions of both TPs and SPs were evaluated separately. These peculiarities, probably reflecting host-specific features of selective constraints and their continuous modulation, are documented by the dynamics of Ka/Ks ratios of hypervariable env domains.

Base Sequence↗
PubMed1999

A combination of nucleoside analogues and a protease inhibitor reduces HIV-1 RNA levels in semen: implications for sexual transmission of HIV infection.

Direct contact with semen is the major route of sexual acquisition of human immunodeficiency virus (HIV) in homosexual and heterosexual partners of seropositive men. In this study, we show that concentrations of HIV-1 RNA molecules in plasma and semen of seropositive patients are related to the duration and type of antiretroviral agents used in treatment. In patients treated with zidovudine alone, 1, 3 and 6 months after the start of therapy, the mean HIV-1 load in plasma was reduced by 0.57, 0.38 and 0.21 log10 and in semen by 0.66, 0.50 and 0.15 log10, respectively. In patients treated with zidovudine plus didanosine at months 1, 3 and 6, the mean decrease in plasma HIV-1 RNA was 1.40, 1.25 and 1.12 log10 and in semen 1.10, 1.41 and 1.32 log10, respectively. In patients treated with a combination of a protease inhibitor and two nucleoside analogues the mean log10 decrease was 1.77, 1.83, 1.71 and 2.38 log10 in plasma and 1.17, 1.74, 2.19 and 3.02 log10 in semen at 1, 2, 3 and 4 months, respectively. Treatment with a combination of a protease inhibitor and two nucleoside analogues caused a dramatic decrease in cell-free HIV-1 RNA in semen, which is a reliable measure of viral load. These findings could have implications for the sexual transmission of HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Rare mutations in a domain crucial for V3-loop structure prevail in replicating HIV from long-term non-progressors.

OBJECTIVE: To evaluate the role of the selective forces exerted by the host on the HIV-1 structures involved in viral entry. DESIGN AND METHODS: The V3 region of the env gene was analysed in cell-free HIV-1 RNA from 17 infected subjects: 11 long-term non-progressors (LTNP) and six symptomless, typical progressor patients. To evaluate the potential biological significance of one of the rare variants detected in the LTNP, it was reproduced by recombinant PCR into a HIV-1 molecular clone. RESULTS: The intrapatient divergence of the V3-loop sequences averaged 8.62% in LTNP and 5.29% in progressors, although LTNP displayed lower divergence from the clade B consensus than progressors (16.65 and 19.76%, respectively). The analysis of non-synonymous and synonymous substitutions indicated that selective pressure was exerted in this region in both LTNP and progressors. Individual peculiarities (unique and rare V3-loop variants) emerged, however, in most sequences from LTNP, and variants bearing mutations in a domain crucial for the V3-loop structure were more prevalent in LTNP (P = 0.0012). The pNL4-3-derived mutant reproducing a V3-loop variant detected in a LTNP was efficiently expressed upon transfection, but the mutant virus was nearly completely unable to infect CD4+ cell lines, activated primary peripheral blood lymphocytes, or monocyte-derived macrophages, suggesting that a defect impaired the entry phase of the replication cycle. CONCLUSIONS: The results indicate that host factors impose selective constraints on the evolution of the HIV-1 structures involved in viral entry. In LTNP, these factors are likely to force the virus into attenuated variants.

Amino Acid Sequence↗
PubMed1998

Early increase of CD4+ CD45RA+ and CD4+ CD95- cells with conserved repertoire induced by anti-retroviral therapy in HIV-infected patients.

Administration of anti-retroviral drugs induces a decrease of viral load associated with increase of CD4+ cell count in most HIV-infected patients. To investigate the early changes in CD4+ cell phenotype induced by anti-retroviral therapy, six patients with CD4+ cell count > 100/mm3 and never treated with anti-HIV therapy were enrolled and blood samples collected several times within 14 days from the initiation of therapy with Zidovudine plus Didanosine. CD4+ cell count and HIV viraemia were investigated at each time point, as well as the expression of CD45RA, CD45RO and CD95/Fas molecules on CD4+ cells, and the T cell receptor (TCR) Vbeta repertoire of CD4+ cells. All patients showed a rapid and dramatic decrease in viral load with a corresponding increase of CD4+ cell count. The main remodelling of CD4+ cell subpopulations took place in the first 14 days of therapy, and consisted of: (i) increased CD4+CD45RA+/CD4+CD45RO+ ratio; (ii) decrease of CD95/Fas expression. The rise in absolute number of CD4+CD45RA+ cells was paralleled by an increase of CD4+CD95/Fas- cells and accounted for most of the early increment of CD4+ cell count. The TCR Vbeta repertoire of CD4+ cells was conserved after anti-HIV therapy, with the exception of two patients with expanded CD4+Vbeta12+ cells, which also tested CD45RA+ and CD95/Fas-. These experiments show that newcomer CD4+ lymphocytes are CD45RA+CD95/Fas- cells, suggesting that blocking HIV replication causes an early and antigen-independent proliferation of possibly 'naive' cells unprimed for CD95/Fas-mediated apoptosis. These cells expressed a conserved and widespread TCR repertoire, suggesting that their capability for antigenic recognition is intact.

Adult↗
PubMed1998

Clinical implications of HIV dynamics and drug resistance in macrophages.

Macrophages are widely recognized as the second major target of HIV in the body. The cellular characteristics of such resting cells markedly affect the dynamics of virus lifecycle, that is slower but far more prolonged that in lymphocytes. In addition, the limited concentrations of endogenous nucleotide pools in macrophages downregulate the enzymatic activity of reverse transcriptase. As a consequence, both the anti-HIV activity and the development of resistance to antiviral drugs in macrophages are substantially different than those found in activated lymphocytes. These peculiar characteristics of virus replication and efficacy of antiviral drugs in macrophages have a natural in vivo counterpart in extralymphoid tissues, where macrophages account for the majority of cells infected by HIV. Furthermore, the replication of HIV in macrophages of testis and central nervous system is far less affected by antiviral drugs than in lymph nodes, because of the presence of natural barriers that markedly diminish the concentration of such drugs. For all these reasons, HIV infection of macrophages should be taken into account in therapeutic strategies aimed to achieve an optimal therapeutic effect in all tissue compartments where the virus hides and replicates.

Anti-HIV Agents↗
PubMed1997

Comparable biological and molecular determinants in HIV type 1-infected long-term nonprogressors and recently infected individuals.

Human immunodeficiency virus type 1 (HIV-1) isolability, rate of replication, phenotype, plasma viremia, and specific intracellular transcripts were cross-sectionally analyzed in 61 HIV-1-seropositive individuals to evaluate the correlations between the virological and molecular correlates of protection and progression in different clinical subsets: recently infected subjects (RIs), long-term nonprogressors (LTNPs), late progressors (LPs), and typical progressors (TPs). Comparison of the major virological and molecular features of HIV-1 infection has defined distinct profiles for different subsets of patients. LTNPs or RIs, as well as LPs or TPs, exhibited similar titers of coculture p24 antigen; the differences between the former and the latter were statistically significant at all the time points tested (p = 0.0001; 0.0003 and 0.0001). Whereas LTNPs and RIs revealed comparable low levels of indexes of viral replication, LPs and TPs showed higher genome and mRNA copy numbers (p = 0.0004 and p = 0.0008, respectively). We demonstrated close biological and molecular similarities between RIs and LTNPs on the one hand, and LPs and TPs on the other. In LTNPs both viral biological properties and viral load are important determinants of the course of the disease.

CD4 Lymphocyte Count↗
PubMed1997

Hemophilia and nonprogressing human immunodeficiency virus type 1 infection.

Seven of 112 hemophiliacs infected with human immunodeficiency virus type-1 (HIV-1) before 1986 through contaminated plasma products are currently healthy, with CD4 T-cell counts above 500 cells/microL, and have never received antiretroviral therapy (long-term nonprogressors [LTNPs]). Seven age and sex-matched hemophiliacs infected in the same period but who have progressive HIV disease (progressors) and one additional slow-progressing individual were also studied. One hundred-fold, 20-fold, and 10-fold lower levels of full-length HIV RNA in plasma, peripheral blood mononuclear cells (PBMCs), and proviral DNA in PBMCs, respectively, were found in LTNPs compared with progressors. Plasma and cell-associated HIV RNA and proviral DNA were lower in LTNPs who tested negative for viral isolation from PBMCs or who were positive only after removal of CD8+ cells. No substantial differences were observed in the in vitro production of chemokines including RANTES, MIP-1 alpha, MIP-1 beta, MCP-1, and interleukin-8 (IL-8) in supernatants of activated PBMCs or CD8-depleted PBMCs of LTNPs, even when HIV isolation was simultaneously accomplished exclusively after removal of CD8+ cells. Low levels of HIV load and replication in peripheral blood are the strongest correlates of nonprogression in this small number of infected hemophiliacs.

Adolescent↗
PubMed1997

Patterns of in vitro anti-human immunodeficiency virus type 1 antibody production in long-term nonprogressors.

With the aim of evaluating the specific pattern of in vitro antibody production (IVAP) in human immunodeficiency virus type 1 (HIV-1)-infected long-term non-progressors (LTNPs), we tested 20 subjects who had remained asymptomatic for more than 8 years with a CD4+ cell count higher than 500/microliter and 59 patients at different stages of HIV-1 infection as controls. In cell cultures, IVAP was detected in 14 out of 20 LTNPs (70%), in 5 out of 6 recent seroconverters (83%), and in all the other control patients. Anti-p24 antibody production was significantly lower in LTNPs than in asymptomatic patients with a more recent infection. Recent seroconverters and patients with AIDS did not produce anti-p24 antibodies (P = 0.02). Anti-gp160 antibodies were produced by peripheral blood mononuclear cells from LTNPs in 12/20 cases. CD4+ cell count was significantly higher in IVAP-negative than in IVAP-positive LTNPs (P = 0.013), while the viral load was not significantly different. Specific anti-HIV-1 antibody production did not seem to be a correlate of long-term nonprogression.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Dynamic features of human immunodeficiency virus type 1 (HIV-1) viremia: kinetics of cell-free HIV-1 RNA after therapeutic plasma exchange.

To gain insight into the variables that influence the dynamics of human immunodeficiency virus type 1 (HIV-1) viremia levels, HIV-1 RNA molecules were quantified in plasma from an infected patient undergoing therapeutic plasma exchange (TPEx). After each TPEx procedure (2000 mL of fluid exchanged per session), HIV-1 genome molecule levels dropped to 58%-63% of the basal level but rapidly reverted to pre-TPEx values (doubling time = 3.50-4.04 h). Of interest, mobilization of extravascular cell-free virions (on average, 5.15 x 10(4) viral genome molecules/h) had already occurred during TPEx. After three daily TPEx procedures, HIV-1 viremia rebounded to basal values, while HIV-1 proviruses and viral transcripts in peripheral blood lymphocytes constantly tested at stable levels. Overall, this study extends previous analyses of the rate of HIV-1 turnover, using an alternative approach to the use of antiretroviral drugs, and it provides, albeit indirectly, insights into the amount and dynamic features of extravascular cell-free virus.

Cell-Free System↗